Identification of novel monocistronic HTLV-1 mRNAs encoding functional Rex isoforms
© Rende et al. 2015
Received: 20 January 2015
Accepted: 19 June 2015
Published: 2 July 2015
Human T cell leukemia virus type 1 (HTLV-1) gene expression is controlled by the key regulatory proteins Tax and Rex. The concerted action of these proteins results in a two-phase kinetics of viral expression that depends on a time delay between their action. However, it is difficult to explain this delay, as Tax and Rex are produced from the same mRNA. In the present study we investigated whether HTLV-1 may produce novel mRNA species capable of expressing Rex and Tax independently.
Results revealed the expression of three alternatively spliced transcripts coding for novel Rex isoforms in infected cell lines and in primary samples from infected patients. One mRNA coded for a Tax isoform and a Rex isoform, and two mRNAs coded for Rex isoforms but not Tax. Functional assays showed that these Rex isoforms exhibit activity comparable to canonic Rex. An analysis of the temporal expression of these transcripts upon ex vivo culture of cells from infected patients and cell lines transfected with a molecular clone of HTLV-1 revealed early expression of the dicistronic tax/rex mRNAs followed by the monocistronic mRNAs coding for Rex isoforms.
The production of monocistronic HTLV-1 mRNAs encoding Rex isoforms with comparable activity to canonical Rex, but with distinct timing, would support a prolonged duration of Rex function with gradual loss of Tax, and is consistent with the two-phase expression kinetics. A thorough understanding of these regulatory circuits will shed light on the basis of viral latency and provide groundwork to develop strategies for eradicating persistent infections.
KeywordsHTLV-1 Rex Splicing
Identification and coding potential of novel alternatively spliced HTLV-1 mRNAs
RT-PCR and qPCR primers and probes
RT-PCR and real time PCR primers
TaxRex s: 5′-GTCCGCCGTCTAG^CTTCC-3′ (exon 1^2 SA)
TaxRex as: 5′-CTGGGAAGTGGG^CCATGG-3′ (exon 2^3 SA)
TaxaRexa s: 5′-ACCACCAACACCATGG^GGTTTG-3′ (exon 2^3a SA)
Rexb s: 5′-ACCACCAACACCATGG^CAGGTC-3′ (exon 2^C SA)
Rexc s: 5′-ACCACCAACACCATGG^GTCCTC-3′ (exon 2^Ca SA)
Rex isoforms as: 5′-GAGTCGAGGGATAAGGAAC-3′
Real time probes
TaxRex: 5′ (FAM)-CCCAGTGGATCCCGTGGAG-3′(TAMRA)
Rex isoforms: 5′(FAM)-AAGGCGACTGGTGCCCCATCTCTGGG-3′(TAMRA)
Quantitation of novel alternatively spliced HTLV-1 mRNAs
Intracellular localization of Rex protein isoforms
Functional analysis of Rex protein isoforms
The finding that Rexb and Rexc show a functional activity comparable to that of Rex and Rexa suggests that their prevalent cytoplasmic localization is likely to result from increased nuclear export or decreased nuclear retention rather than an intrinsic defect in nuclear import, which would be expected to seriously impinge on their function.
The amino-acid changes in Rexb and Rexc do not affect the sequence of the nuclear localization signal (NLS) or the leucine-rich nuclear export signal/activation domain (NES/AD). However, the presence of 25 or 24 extra amino-acids in Rexb and Rexc respectively, immediately downstream of the NLS and 55 amino-acids upstream of the NES/AD (Figure 2) might induce conformational changes affecting the function of the NLS and/or the NES/AD. It is also possible that the extra residues affect the protein phosphorylation status, which is known to be important for Rex function , as well as subcellular localization in the case of Rex of HTLV-2 .
Distinct temporal regulation of mRNAs encoding Rex isoforms
These findings provide insight into the mechanisms controlling HTLV-1 expression and suggest that the production of alternatively spliced monocistronic mRNAs coding for Rex isoforms contributes to the two-phase kinetics of HTLV-1 expression. The resulting pattern of viral gene expression might be important to temporally restrain the expression of highly immunogenic viral epitopes (e.g. Tax, Gag, Env), thus favouring escape from the immune response and establishment of long term persistence in the host, a key feature of HTLV-1 infection. A better understanding of how these regulatory circuits are established and maintained will shed light on the basis of viral latency and may provide groundwork for the development of new therapies for eradicating persistent infections.
FR carried out the molecular cloning, immunofluorescence, immunoblot and functional assays, analyzed and interpreted the data, IC carried out the qRT-PCR assays, analyzed and interpreted the data. VA and VWV helped with in initial pilot experiments. VC and GF conceived the study, analyzed and interpreted the data, and DMD participated in its design and coordination and helped to interpret the results. All authors read and approved the final manuscript.
We thank Luigi Chieco-Bianchi for discussions and Charles Bangham and Graham Taylor for discussions and patient samples. The research was supported by investigator Grants from the Associazione Italiana per la Ricerca sul Cancro (AIRC; nos. 4175 and 13378), an AIRC-Cariverona Regional Grant, and the University of Padua (Ateneo Grant no. CPDA124913/12).
Compliance with ethical guidelines
Competing interests The authors declare that they have no competing interests.
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